Hypoxia of the retina has been associated with the initiation and progression of blinding retinal vascular diseases. For example, age-related macular degeneration (AMD), retinopathy of prematurity (ROP),1,2 proliferative diabetic retinopathy (PDR),3 and retinal vein occlusion (RVO)4 are blinding conditions with neovascular components that develop from ischemia-induced retinal hypoxia. Typically, retinal hypoxia activates the transcription of hypoxia-regulated pro-angiogenic growth factors/cytokines such as vascular endothelial cell growth factor (VEGF)9 and angiopoietin-like protein-4 (ANGPTL4).10 These factors elicit a neovascular response that manifests in the formation of pre-retinal neovascular structures that enhance morbidity, often leading to blindness in individuals afflicted with ROP, PDR, and RVO.
More specifically, in ROP, ischemia arises from attenuated physiologic blood vessel development in preterm infants receiving supplemental oxygen to compensate for under-developed lung function.5,6 When the oxygen therapy is discontinued and the infant is placed in normoxia, the peripheral retina is avascular (ischemia), and becomes hypoxic7. Hyperglycemia and hyperlipidemia are causally linked to capillary dropout and vasoregression in the diabetic retina, leading to focal avascularity (ischemia) and incipient retinal hypoxia that triggers the onset of PDR. Similarly, in branch RVO, injury or atherosclerosis results in the formation of an occlusive thrombus, reducing blood flow (ischemia) initiating the development of retinal hypoxia.8 
In view thereof, several analytical platforms have been applied to measuring retinal oxygen pressure (PO2) levels including, but not limited to, the use of oxygen sensitive electrodes,11 nuclear magnetic resonance (NMR),12 retinal oximetry,13 oxygen-dependent molecular phosphorescence quenching,14 doppler optical coherence tomography (D-OCT).15 Oxygen electrodes permit the acquisition of reliable data but are invasive and cannot be used for rodents due to their small globes. NMR is minimally invasive, however it is not a direct measure of oxygen tension and the resolution is appreciably less than optical methods.18-20 Retinal oximetry and doppler OCT are methods that hinge on the differences in the spectral characteristics of oxyhemoglobin and hemoglobin in the intravascular compartment, and their relative abundance in arteries compared to veins. These measurements may be performed in living systems, however, they are indirect and mathematical modeling is required to estimate the perivascular oxygen pressure. Phosphorescent quenching relies on intravascular oxygen levels providing only limited assessment of the oxygen pressure in the retinal tissue.
Other methods that have been applied to measuring PO2 include visible-light optical coherence tomography (vis-OCT)16 and immunohistochemical analysis.17 For example, immunohistochemical analysis may include pimonidazole-mediated immunohistochemistry. While pimonidazole-mediated immunohistochemistry is a common method to study retinal hypoxia, the technique is limited by its exclusively ex vivo method of examination.21,22 With regard to vis-OCT, although a number of methods have been reported in the literature to visualize tumor hypoxia using positron emission tomography, none of these methods have been applied to the detection of retinal hypoxia. Additionally, these vis-OCT methods carry the risks associated with use of short-lived isotopes. For these and other reasons, the techniques discussed above are not currently available or not suitable for measuring retinal hypoxia in living animals in real time.
In an attempt to address these issues, the instant inventors previously described the development of HYPDX-1, HYPDX-2, and HYPDX-3 as sensitive fluorophore-labeled imaging probes to detect hypoxia.23,24 These fluorescent probes are reduced by nitroreductases or azoreductases, facilitating their retention within hypoxic cells of the retina, allowing ex vivo hypoxia detection.25 However, their application to in vivo imaging is limited due to poor pharmacokinetic parameters.
Accordingly, the currently available methods fail to provide noninvasive imaging techniques capable of detecting and monitoring retinal hypoxia in living systems. Thus, there is a need for improved compositions and methods that would provide in vivo hypoxia imaging. The presently disclosed embodiments fulfill such a need, and offer other related advantages.